DETECTION OF RARE RNA SEQUENCES BY SINGLE-ENZYME IN-SITU REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION - HIGH-RESOLUTION ANALYSES OF INTERLEUKIN-6 MESSENGER-RNA IN PARAFFIN SECTIONS OF LYMPH-NODES

To study the distribution pattern of interleukin-6 (IL-6)-producing ce lls ill normal human lymph nodes, we applied the in situ reverse trans cription-polymerase chain reaction technique. We describe a new modifi cation of this technique for monitoring small amounts of specific nucl eotide sequences in conventional paraffin sections. This technique dif fers In at least two respects from those described earlier The two dec isive steps are: I) the reverse transcription of mRNA and the subseque nt amplification of cDNA by polymerase chain reaction are performed by a new single enzyme capable of both reaction types in one and the sam e medium without buffer exchange; and 2) for the specific detection of the amplified cDNA, a modified version of the primed in situ labeling technique tons used. The technique, carried out on normal human lymph nones, traces a low load of IL-6 mRNA in fibroblasts, endothelial cel ls, and a minor population of T lymphocytes in the pulp region. High l evels of expression were encountered in about 20% of perisinusoidal pu lp macrophages. In addition, moderate activity was detectable in sinus lining cells, Because no major activity was found in the germinal cen ters of the lymphoid B follicles and in the T zone, it is suggested th at the plasma cell differentiation ensuing from primary and secondary B-cell immunization is mainly effected by the sinus lining cells as we ll as perifollicular and perisinusoidal pulp macrophages capable of pr oducing high amounts of IL-6.